1. Field of the Invention
This invention relates to novel microorganisms and vaccines for the protection of aquatic animals from bacterial diseases such as, motile Aeromonas septicemia, enteric septicemia, and streptococcal diseases. This invention also relates to methods for using the novel vaccines to protect aquatic animals from bacterial disease as well as to methods for preparing the vaccines using novel antibiotic resistance strategies.
2. Description of the Related Art
Disease problems constitute the largest single cause of economic losses in aquaculture and bacterial infections constitute the most important source of disease problems in various types of production (Meyer, 1991, J. Anim Sci. Volume 69, 4201-4208). Estimated economic impact from infections caused by bacterial infection on aquaculture industry annually is at least $10 million in the USA alone and more than $100 million globally (Shoemaker et al., 2010. J. Fish Dis. Volume 33, 537-44).
Antibiotics and chemotherapeutic drugs have been used for disease management in feed additives and in direct administration into fish pond water; however, there has been an increase in drug resistant strains (Son et al., 1997, Letters in Appl. Microbiol., Volume 24, 479-482); (Harikrishnan and Balasundaram, 2005, Reviews in Fisheries Science, Volume 13, 281-320).
Edwardsiella ictaluri (E. ictaluri) causes enteric septicemia of catfish (ESC) which is the most prevalent disease affecting farm-raised catfish, Ictalurus punctatus. E. ictaluri is a facultative Gram-negative flagellated bacterium akin to phylogenetically related Salmonella (Thune et al., J. World Aqua Soci, Volume 28, 193-201, 1997; Zhang and Arias, Syst. Appl. Microbiol., Volume 30, 93-101, 2007). ESC is responsible for approximately $20 to $30 million in annual losses to catfish farmers in the southeastern United States (Plumb and Vinitnantharat, J. Fish. Dis., Volume 16, 65-71, 1993). ESC is generally an acute septicemia that develops very quickly, resulting in heavy mortalities at as early as 4 days after infection (Thune et al., supra; Newton et al., J. Fish. Dis., Volume 12, 335-347,1989; Wolters and Johnson, J. Aquat. Anim. Health, Volume 6, 329-334, 1994).
Streptococcus iniae is a significant fish pathogen impacting aquaculture production worldwide. Since its original isolation in 1976 from Amazon freshwater dolphin (Inia geoffrensiss) (Pier and Madin, 1976, Inyt. J. Syst. Bacteriol, Volume 26, 545-553), Streptococcus iniae has become a major aetiological agent of Streptococcus in farmed and wild finfish worldwide, affecting more than 30 species of fish, including trout, yellowtail, tilapia, barramundi, and hybrid striped bass (Cheng et al, 2010, Vaccine Volume 28, 2636-2641; Eyngor et al., 2008, Appl. Environ Microbiol., Volume 74, 6892-6897; Agnew and Barnes, 2007, Vet. Mcirobiol., Volume 122, 1-15; Ferguson et al., 2000, Vet Rec Volume 147, 662-664; Barnes, 2007, Vet Microbiol., Volume 122, 1-15; Ferguson et al., 2000, Vet Rec., Volume 147, 662-664; Bromage et al., 1999, Dis Aquat Organ, Volume 36, 177-181; Eldar et al., 1999, Dis Aquat Organ, Volume 36, 121-127). More recently, this bacterium has also been identified as a potential zoonotic pathogen, with at least 25 cases of human infection by S. iniae confirmed to date (Agnew and Barnes, 2007, supra; Sun et al., 2007, J. Med. Microbiol., Volume 56, 1246-1249; Koh et al., 2004, Emerg Infect Dis, Volume 10, 1694-1696; Weinstein et al., 1997, N. Engl J. Med., Volume 337, 589-594).
Edwardsiella tarda is a Gram-negative, motile, rod-shaped, aquatic bacterial pathogen which is highly infectious in both warm and cold water fish species. The bacterium is commonly encountered in channel catfish ponds, intensive tilapia culture, and eel production systems and is, therefore, a constant threat of disease. In the channel catfish (Ictalurus punctatus), E. tarda, the causative agent of enteric septicemia disease, has been isolated from channel catfish in areas of the southeastern United States. The disease also affects numerous other cultured fish species, sport fish, such as large-mouth bass, baitfish, and aquarium fishes. Wyatt et al. (Applied Environmental Microbiology, Volume 38, 710-714, 1979) found that in E. tarda positive catfish ponds, this bacterium was isolated from 75% of the pond water, 64% of pond mud, and 100% of apparently healthy frog, turtle, and crayfish samples. Meyer and Bullock (Applied Microbiology, Volume 25, 155-156, 1973) highlighted the food safety problem of E. tarda when they reported that 88% of dressed catfish were culture-positive for E. tarda. This usually results in a shut down of the processing lines until they are cleaned and disinfected because of the potential risk of human infection.
Streptococcus agalactiae is a Group B streptococcal bacterium that causes severe economic losses in a number of species of cultured and wild fish. This infectious bacterium is common in aquaculture facilities where fish are intensively cultured in fresh, brackish, or marine waters. The high densities of fish and the aqueous environment favor the rapid transmission of streptococcal disease. Moreover, infected cultured fish may transmit the disease to wild fish populations or infected wild fish may transmit the disease to cultured popultations.
Aeromonas hydrophila infection results in hemorrhagic septicemia and heavy mortalities in cultured and wild fish. There is no product that has been licensed for use against the motile aeromonads within the United States (Cipiano, R. C., 2001, Revision of Fish Disease Leaflet 68, U.S. Dept. Interior, Fish and Wildlife Service Div. of Fishery Res., Washington, D.C.).
Methods to control diseases in fish include the use of chemical therapeutics such as antibiotic-medicated food (Darwish 2007, Journal of Aquatic Animal Health, Volume 19, 1-7). However, large scale use of antibiotics in aquaculture is expensive and usually ineffective because sick fish normally do not eat. Furthermore, fish have developed resistance to approved food fish antibiotics, such as oxytetracycline, florfenical, and ormethorprimsulphamethoxine (Eldar et al., Dis. Aquat. Organ., Volume 36, 121-127, 1999; Sun et al, J. Med. Microbiol., Volume 56, 1246-1249, 2007; Tu et al., 2008, Microb Drug Resist. Volume 14, 311-316; Nawaz et al., 2006, Appl. Environ. Microbiol., Volume 72, 6461-6466; Balotescu et al., 2003, Roum. Arch. Microbiol. Immunol., Volume 62, 179-189; Hatha et al., 2005, Int. J. Food Microbiol., Volume 98, 131-134,; Saavedra et al., 2004, Int. Microbiol., Volume 7, 207-211.)
Alternative methods to control fish diseases include the use of vaccines. The most extensively studied Streptococcus iniae vaccines are killed bacterins consisting of formalin killed bacteria cells of pathogenic Streptococcus iniae strains (Eldar et al, 1997, Vet. Immunol. Immunopathol., Volume 56, 175-183; Bercovier et al., 1997, Dev. Biol. Stand., Volume 90, 153-160). These formalin killed bacteria cells of Streptococcus iniae have been previously successfully used as vaccines to protect rainbow trout in Israel. However, recently, it has been reported that these killed vaccines are unable to protect fish from infection by other isolates (serotypes) of Streptococcus iniae (Bachrach et al, 2001, Appl Environ Microbiol, Volume 67, 3756-3758; Eyngor et al., 2008, Appl Environ Microbiol, Volume 74, 6892-6897). In addition to killed vaccines, live attenuated S. iniae strains defective in phosphglucomutase and M-like protein have been reported to offer protection against homologous S. iniae challenge (Locke et al., PLoS One, Volume 3, c2824, 2008; Buchanan et al., Infect. Immun., Volume 73, 6935-6944, 2005). However, it is not clear whether they offer protection against heterologous S. iniae. 
Attenuated live bacterial vaccines such as rifampicin-resistant Edwardsiella ictaluri (AquaVac-ESC) and Flavobacterium columnare (AquaVac-COL), both licensed to Intervet/Shering-Plough, have been developed through a rifampicin-resistant strategy (Klesius and Shoemaker, 1999, Adv. Vet. Med., Volume 41, 523-537; Shoemaker et al., 2007, Vaccine, Volume 25, 1126-1131) and offer great protection against infections by E. ictaluri and F. columnare. Rifampicin works by inhibiting DNA-dependent RNA polymerase in bacterial cells by binding its beta-subunit, thus preventing transcription to RNA and subsequent translation to proteins (Schullz and Zillig, 1981, Nucleic Acids Res., Volume 9, 689-6906). This strategy relies on the ability of rifampicin to induce the appearance of rough mutants on relatively solid culture media, i.e., agar plates. It has been demonstrated that the rifampicin-resistant RE-33 strain of E. ictaluri was unable to cause ESC, but was able to stimulate protective immunity in catfish (Klesius and Shoemaker, Adv. Vet. Med., Volume 41, 523-537, 1999). However, it is not clear whether other antibiotics could also be used to attenuate bacteria for the purpose of novel vaccine development.
Klesius et al. (US Patent Application Publication 2010/0221286, Sep. 2, 2010) discloses a modified live rifampicin-resistant Aeromonas hydrophila vaccine for aquatic animals wherein the Aeromonas hydrophila mutants were modified by using a low initial concentration of 2.5 μg/ml rifampicin and ending at about 320 μg/ml of rifampicin after 44 passages of bacteria on relatively solid culture media, i.e., agar plates. The mutants obtained by this method were used to vaccinate fish either by intraperitoneal (IP) injection or bath immersion.
Klesius et al. (U.S. Pat. Nos. 6,019,981, Feb. 1, 2000 and 6,153,202, Nov. 28, 2000) disclose modified live rifampicin-resistant Edwardsiella ictaluri vaccines for aquatic animals wherein the E. ictaluri mutants were modified by using a low initial concentration of 5.0 μg/ml rifampicin and ending at about 320 μg/ml of rifampicin after 44 passages of bacteria on relatively solid culture media, i.e., agar plates. The mutants obtained by this method were used to vaccinate fish both post-hatch and in ovo using bath immersion.
Evans et al. (U.S. Pat. No. 7,067,122) disclose a rifampicin-resistant live vaccine against Edwardsiella tarda. E. tarda was grown on modified tryptic soy agar (TSA) plates in increasing concentrations of rifampicin starting at 10 μg/ml and ending at 320 μg/ml, increasing at 20 μg/ml increments.
Evans et al. (U.S. Pat. No. 7,204,993, Apr. 17, 2007) disclose a Streptococcus agalactiae vaccine prepared with killed cells of isolated β-hemolytic Streptococcus agalactiae. 
Novobiocin, also known as albamycin or cathomycin, is a natural antibiotic produced by the actinomycete Streptomyces niveus, a member of the order of Actinobacteria (Kominek, 1972, Antimicrob. Agents Chemother., Volume 1, 123-134,). Novobiocin works as a natural inhibitor of bacterial DNA gyrase, resulting in bacterial cell-death (Gellert et al., 1976, Proc. Natl. Acad. Sci. USA, Volume 73, 4474-4478). DNA gyrase, an ATP-dependent enzyme that acts by creating a transient double-stranded DNA break, is essential for efficient DNA replication, transcription, and recombination by catalyzing the negative supercoiling of DNA (Mdluli and Ma, 2007, Infec. Disord. Drug Targets, Volume 7, 159-168).
While various vaccines have been developed that are effective for Aeromonas hydrophila, Edwardsiella ictaluri, Edwardsiella tarda, Streptococcus agalactiae and Streptococcus iniae infections of aquatic animals, there remains a need in the art for efficacious and safe vaccines for the aquaculture industry. The present invention described below includes attenuated live vaccines and provides methods for treating aquatic animals using said vaccines as well as methods for preparing live attenuated bacterial vaccines that are efficacious and safe and different from related art vaccines and methods.